Comparison of Steroid Hormone Hydroxylations by and Docking to Human Cytochromes P450 3A4 and 3A5

J Pharm Pharm Sci (www.cspsCanada.org) 22, 332 - 339, 2019

Toshiro Niwa1,*, Kanae Narita1, Ayaka Okamoto1, Norie Murayama2, and Hiroshi Yamazaki2

1 School of Pharmacy, Shujitsu University, 1-6-1 Nishigawara, Naka-ku, Okayama 703-8516, Japan. 2 Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165, Higashi-Tamagawa Gakuen, Machida, Tokyo 194-8583, Japan

Received, May 13, 2019; Revised, July 18, 2019; Accepted, July 19, 2019; Published, July 21, 2019.

ABSTRACT - Purpose: Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (P450 or CYP) 3A4 and CYP3A5 and their molecular docking energy values were compared to understand the catalytic properties of the major forms of human CYP3A, namely, CYP3A4 and CYP3A5. Methods: Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4 and CYP3A5 were determined by liquid chromatography. Docking simulations of these substrates to the heme moiety of reported crystal structures of CYP3A4 (Protein Data

Bank code ITQN) and CYP3A5 (6MJM) were conducted. Results: Michaelis constants (Km) for CYP3A5- mediated 6β-hydroxylation of testosterone and progesterone were approximately twice those for CYP3A4, whereas the value for cortisol 6β-hydroxylation mediated by CYP3A5 was similar to the value for that by

CYP3A4. Maximal velocities (Vmax) of the three steroid hormones 6β-hydroxylation catalyzed by CYP3A5 were 30%-63% of those by CYP3A4. Thus, Vmax/ Km values of these hormones for CYP3A5 resulted in 22%- 31% of those for CYP3A4. The differences in the docking energies between CYP3A4 and CYP3A5 for steroid hormones were slightly correlated to the logarithm of CYP3A5/CYP3A4 ratios for Km values (substrate affinity). Conclusions: The Vmax, rather than Km values, for CYP3A5-mediated 6β-hydroxylation of three steroid hormones were different from those for CYP3A4. Molecular docking simulations could partially explain the differences in the accessibility of substrates to the heme moiety of human CYP3A molecules, resulting in the enzymatic affinity of CYP3A4 and CYP3A5. ______

INTRODUCTION differences in catalytic properties have been reported (5, 6). We previously reviewed the reported values

Cytochrome P450 (P450 or CYP) 3A is the most for (a) Michaelis-Menten constants (Km), maximal important human P450 subfamily, due to its high velocities (Vmax), and intrinsic clearance (CLint, abundance in the human hepatic microsomes, which Vmax/Km) values for reactions mediated by CYP3A4 accounts for approximately 30% of total P450s and/or CYP3A5, (b) inhibition constants (Ki) and enzymes (1, 2). Furthermore, CYP3A enzymes are 50% inhibitory concentrations (IC50), and (c) responsible for 50% of all pharmaceuticals oxidation maximum inactivation rate constants (kinact), and (3). Inducible CYP3A4 is generally recognized as demonstrated that the substrate specificities of the predominant CYP3A form expressed in human CYP3A4 and CYP3A5 generally overlap, but some livers and intestines. On the other hand, differences can be identified. (7, 8). polymorphically expressed CYP3A5 contributes as It is well established that some steroid hormones much as 50% of hepatic CYP3A in a third of are metabolized by not only steroidogenic P450s, Caucasians and about half of African–Americans (4). such as CYP11B1, CYP11B2, CYP17A1, CYP19A1, It was thought that the substrate specificity of ______CYP3A5 is similar to that of CYP3A4, because Corresponding Author: Prof. Toshiro Niwa, PhD, School of CYP3A4 and CYP3A5 are 83% homologous in Pharmacy, Shujitsu University , 1-6-1 Nishigawara, Naka-ku, terms of amino acid sequences. However, some Okayama 703-8516, Japan; E-mail: [email protected]

332 J Pharm Pharm Sci (www.cspsCanada.org) 22, 332 - 339, 2019

CYP27A1, but also by drug-metabolizing P450s MATERIALS AND METHODS including CYP2C, CYP2D, and the CYP3A subfamily (9, 10). In terms of hydroxylation at the Materials C6β position, testosterone 6β-hydroxylation is CYP3A4 and CYP3A5 expressed in recombinant predominantly catalyzed by the CYP3A subfamily Escherichia coli (Bactosomes) were obtained from (9, 10) and is recommended as a preferred reaction Cypex Ltd (Dundee, UK). Testosterone and cortisol of CYP3A4/5 for in vitro experiments in the were purchased from Nakarai Tesque (Kyoto, Japan) guidelines for new drug applications regarding in and Fujifilm Wako Pure Chemicals (Osaka, Japan), vitro studies of drug metabolism and drug–drug respectively. Metabolites 6β-hydroxytestosterone interactions regulated by the US Food and Drug and 6β-hydroxycortisol were obtained from Sigma- Administration (FDA), European Medicines Agency Aldrich (St Louis, MO, USA). Progesterone and 6β- (EMA), and Japanese Ministry of Health, Labour hydroxyprogesterone were purchased from Tokyo and Welfare (11-13). The ratio of 6β-hydroxycortisol Chemical Industry (Tokyo, Japan) and Steraloids Inc. to cortisol, a stress hormone, in plasma or urine has (Newport, RI USA), respectively. All other reagents been used as an in vivo endogenous marker of and organic solvents used were of the highest purity CYP3A4/5 metabolic activity (14-16). Progesterone commercially available. is hydroxylated by drug-metabolizing P450s including CYP2D6 and CYP3A4 at the C2β, C6α, Hydroxylation Activities of Steroid Hormones C6β, C16α, and C21 positions, as well as by the Testosterone and progesterone 6β-hydroxylation steroidogenic CYP21A1 at the C21 position (9, 10). activities were determined as described previously However, there are few reports that compare, in with minor modification (19-22). The incubation detail, the kinetic parameters for the hydroxylation mixtures consisted of 10 pmol/mL CYP3A4 or of these steroid hormones mediated by CYP3A4 and CYP3A5, 1 mM NADPH, and 100 mM potassium CYP3A5. phosphate buffer (pH 7.4) in a final volume of 0.5 We previously conducted docking simulation mL. Incubation times were 5 min, with the exception studies for substrates and inhibitors of P450 proteins, of cortisol 6β-hydroxylation by CYP3A4 (10 min) including CYP3A4 and CYP3A5, and demonstrated or CYP3A5 (20 min). For the 6β-hydroxycortisol that some of the results were consistent with the assay, the reaction was terminated by adding 3.5 mL enzymatic characteristics observed in in vitro studies, of ethyl acetate. The organic phase (3 mL) was although the primary sequence of CYP3A5 was evaporated and the residue was immediately aligned with that of CYP3A4 using three- dissolved in 200 µL of 5 mM KH2PO4/methanol dimensional modeling software (17, 18). (75:25). HPLC analysis was performed by injecting In the present study, we compared the CYP3A4 50 µL into an Inertsil ODS-3 column (150×4.6 mm and CYP3A5-mediated 6β-hydroxylation of three I.D., GL Sciences Inc.), with the column temperature steroid hormones: testosterone, progesterone, and set at 40 °C. The UV-detector was set at 240 nm. cortisol. In addition, we carried out docking Mobile phase A was 5 mM KH2PO4/methanol simulations for the binding of CYP3A4 and (75:25) and mobile phase B was 5 mM

CYP3A5 with steroid hormones using a docking KH2PO4/methanol (20:80). A gradient elution at a simulation program and compared the results with flow rate of 1mL/min was performed as follows: 0- previously reported compounds (18). We report 15 min 0% B, 15-16 min 0-100% B (linear gradient), herein that the differences in accessibility of 16-25 min 100% B, 25-26 min 100-0% B (linear substrates to the heme of human CYP3A molecules gradient), and 26-40 min 35% B. Calibration curves resulted in the apparent enzymatic affinity of were linear for 6β-hydroxycortisol concentrations CYP3A4 and CYP3A5. ranging from 0.1 to 5 µM. All data were analyzed using the average of triplicate determinations. In the preliminary experiments, the linearity of the reaction with P450

333 J Pharm Pharm Sci (www.cspsCanada.org) 22, 332 - 339, 2019 concentration and incubation time was confirmed for RESULTS each CYP3A subfamily. Km and Vmax values were calculated by performing Michaelis-Menten kinetics The kinetic parameters for the 6β-hydroxylation of using nonlinear least squares regression by means of three steroid hormones by CYP3A4 and CYP3A5 MULTI (23). were estimated by fitting the values into the

Michaelis-Menten plots (Table 1). The Km values for Docking Simulation of Steroid Hormones with CYP3A5-mediated 6β-hydroxylation of testosterone Human P450 Enzymes and progesterone were approximately twice those The crystal structures of CYP3A4 (Protein Data for CYP3A4, whereas the value for cortisol 6β- Bank code ITQN) and CYP3A5 (6MJM) were used hydroxylation by CYP3A5 was similar to that of

(24, 25). Prior to the docking simulations, the CYP3A4. The Vmax values for CYP3A5-mediated energies of the CYP3A4 and CYP3A5 structures 6β-hydroxylation of the three steroid hormones were were minimized using the CHARMM22 force field. 30-63% of those for CYP3A4. Thus, Vmax/ Km values Docking simulations were carried out for the binding for the 6β-hydroxylation of these hormones by of substrates to P450 enzymes using the MMFF94x CYP3A5 were 22-31% of those for CYP3A4. For force field distributed in the Molecular Operating both CYP3A4 and CYP3A5, Km values for Environment (MOE) ASEDock software (Chemical progesterone were lowest, followed by testosterone.

Computing Group, Montreal, Canada) (18). Twenty Vmax values for testosterone were highest among the solutions were generated for each docking three steroid hormones, and Vmax/ Km values for experiment and then ranked according to total cortisol were lowest. interaction energy (U value). Molecular docking simulations were conducted on six steroid hormones with CYP3A4 and CYP3A5

Literature Search for Kinetic Parameters (Table 2). For estrone, the Km value for CYP3A5- For the correlation analysis, kinetic parameters such mediated 4-hydroxylation is reported to be higher as Km, Vmax, and Vmax/Km for several compounds, than that for CYP3A4 (10, 26). As such, ligand-P450 including estradiol and estrone, were obtained from interaction energies (U values) were found to be the literature (10, 18, 26). Correlation analysis was lower for CYP3A4 than for CYP3A5. However, U carried out using Prism (GraphPad Software, La values for CYP3A5 in other steroid hormones were Jolla, CA). lower than those for CYP3A4. In all cases, the docking distance was between 3.82 and 11.3Å. Several compounds, other than these steroid hormones, were investigated in order to confirm the present docking results.

Table 1. Kinetic parameters for steroid 6β-hydroxylation activities by recombinant human CYP3A4 and CYP3A5 Km Vmax Vmax/ Km Steroid CYP3A4/5 (µM) (nmol/min/nmol CYP3A) (mL/min/nmol CYP3A) Testosterone CYP3A4 63.8±10.5 (100) 125±7 (100) 1.96 (100) CYP3A5 119±24 (186) 51.1±4.1 ( 41) 0.43 ( 22) Progesterone CYP3A4 14.3±2.7 (100) 43.4±1.6 (100) 3.03 (100) CYP3A5 29.2±3.6 (204) 27.2±0.8 ( 63) 0.93 (31) Cortisol CYP3A4 358±37 (100) 12.2±0.5 (100) 0.034 (100) CYP3A5 413±136 (115) 3.6±0.5 ( 30) 0.0087 ( 26) Kinetic parameters were determined by nonlinear regression analysis (mean ± standard deviation, n = 7 substrate concentrations in the range of 2–1,000 μM, in triplicate determinations). Values in parentheses are percentage of those for CYP3A4.

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Vincristine, which is a typical substrate of CYP3A5 CYP3A5/CYP3A4 ratios for Vmax/Km values (Fig. and has higher Vmax and Vmax/Km values and a lower 1C). U: interaction energy in kcal/mol. Seven

Km value for reactions mediated by CYP3A5 than by reactions of five steroid hormones as shown in Table CYP3A4 (27), could more closely dock to the heme 2, except for DHEA, were investigated. Dotted lines region of CYP3A5 (10.8Å), with a slightly lower U show the regression curve between the logarithm of value, than to that of CYP3A4 (18.5Å). β- the CYP3A5/CYP3A4 ratios of Km, Vmax, and

Endosulfan, which has a lower Km value and higher Vmax/Km values and the differences between U values

Vmax and Vmax/Km values for reactions mediated by for CYP3A4 and CYP3A5 for steroid hormones. CYP3A5 than by CYP3A4 (28), had a lower U value for CYP3A5 than for CYP3A4. On the other hand, DISCUSSION midazolam 1'- and 4-hydroxylations which have higher Km and Vmax values for reactions catalyzed by Testosterone 6β-hydroxylation is recommended as a CYP3A4 than by CYP3A5 (8, 18), had a lower U preferred reaction of CYP3A4/5 for in vitro value for CYP3A4 than that for CYP3A5. experiments (11-13) and the 6β- We investigated the relationship between the hydroxycortisol/cortisol ratio is used as an in vivo ratios of mean Km, Vmax, and Vmax/Km values obtained endogenous marker of CYP3A4/5 metabolic in the present study and previously reviewed for activities (14-16). In addition, cortisol, a stress CYP3A4 and CYP3A5 (18), and the differences hormone, is known to be one of the many hormones between the U values for these P450s (Fig. 1). The responsible for the physiological changes involved difference in U values between CYP3A5 and in response to stress, as well as a glucocorticoid CYP3A5 were slightly correlated with the logarithm hormone that is produced by the adrenal glands and of CYP3A5/CYP3A4 ratios for Km (r = 0.62, p = released for a variety of reasons (29). Progesterone

0.14, Fig. 1A) and Vmax values (r=0.64, p = 0.12, Fig. 6β-hydroxylation is catalyzed by drug-metabolizing 1B), but not with the logarithm of P450s, including CYP3A4 and CYP2D6 (9, 10).

Table 2. Docking simulation results of steroid hormones with respect to human CYP3A4 and CYP3A5 U value (kcal/mol) Distance (Å) Compound Reaction CYP3A4 CYP3A5 CYP3A4 CYP3A5 Testosterone 6β-Hydroxylation -26.3 -38.5 5.03 7.36 Progesterone 6β-Hydroxylation -11.8 -34.7 10.7 11.3 Cortisol 6β-Hydroxylation -27.0 -44.7 6.65 6.99 DHEA 7β-Hydroxylation -27.6 -39.0 8.31 8.22 16α-Hydroxylation -30.4 -41.8 5.68 6.03 Estradiol 2-Hydroxylation -5.7 (18) -18.8 9.27 7.24 4-Hydroxylation -5.7 (18) -18.8 9.21 7.62 Estrone 2-Hydroxylation -38.0 -20.7 4.04 7.65 4-Hydroxylation -38.0 -20.7 3.82 8.81 Vincristine M1 formation -17.8 (18) -20.6 18.5 10.8 β-Endosulfan Sulfate formation -7.17 (18) -21.1 5.77 9.92 Midazolam 1'-Hydroxylation -52.3 (18) -26.0 4.64 4.71 4-Hydroxylation -52.3 (18) -24.0 5.31 4.52 DHEA: dehydroepiandrosterone.

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(A) Km Although Km and Vmax values for testosterone 6β- hydroxylation by recombinant CYP3A4 and/or 30 CYP3A5 have already been reported as 24-110 µM

20 (for CYP3A4) and 44-400 µM (for CYP3A5) for Km

(CYP3A4) values and 3.8-3600 nmol/min/nmol P450 (for y = 45.2x - 15.7 U 10 r = 0.62, p = 0.14 CYP3A4) and 2.2-1600 nmol/min /P450 (for

0 CYP3A5) for Vmax values (7, 10, 30-36), the values

obtained in the present study (Km: 63.8 µM for (CYP3A5) - (CYP3A5) -10

U CYP3A4 and 119 µM for CYP3A5, Vmax: 125 -20 nmol/min/nmol P450 for CYP3A4 and 51.1 nmol/min/nmol P450 for CYP3A5) were similar to -30 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 the medians of the reported values. In the present

study, Km values for 6β-hydroxylation of three log [Km (CYP3A5) / Km (CYP3A4)] steroid hormones (testosterone, progesterone, and

(B) Vmax cortisol) mediated by CYP3A5 were at most twice

30 those for the CYP3A4-mediated reactions, whereas Vmax values for the hydroxylation catalyzed by 20 CYP3A5 were 30-63% of those for CYP3A4.

(CYP3A4) 10 y = 41.7x + 2.2 Therefore, Vmax/ Km values, which correspond to U r = 0.64, p = 0.12 intrinsic clearance, for the hydroxylation of these 0 hormones by CYP3A5 were 22-31% of those for -10 CYP3A4. These results suggest that testosterone 6β- (CYP3A5) - (CYP3A5) U -20 hydroxylation activity in human liver microsomes for in vitro studies of drug-drug interactions would -30 depend on the CYP3A4 level rather than CYP3A5. -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 Thus, the addition of other substrates, such as log [V (CYP3A5) / V (CYP3A4)] max max midazolam, might be necessary for the assessment

30 PMDA (11, 13). Furthermore, the effect of CYP3A5 polymorphism on the plasma or urine ratio of 6β- 20 hydroxycortisol/cortisol in vivo as a marker for

(CYP3A4) 10 stress might be of minor importance. Further clinical U studies are necessary to elucidate the problem. 0 We previously carried out docking simulations -10 for the binding of CYP3A4 and CYP3A5 with 13 (CYP3A5) - (CYP3A5)

U substrates using docking simulation program MOE -20 and found that the docking energy ratio between -30 CYP3A5 and CYP3A4 were not significantly -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 correlated with the CYP3A5/CYP3A4 ratios of Vmax log [Vmax/Km (CYP3A5) / Vmax/Km (CYP3A4)] and Vmax/Km values, nor with molecular weight, Figure 1. Relationship between the ratios of Km (A), Vmax volume, or log P values of substrates. Furthermore, (B), and Vmax/ Km (C), and the differences between neither the differences in the enzyme source docking energy values for CYP3A4 and CYP3A5. U: (baculovirus infected insect cells or Escherichia coli interaction energy in kcal/mol. Seven reactions of five steroid hormones as shown in Table 2, except for DHEA, engineered to express P450s) nor the were investigated. Dotted lines show the regression curve supplementation or co-expression of cytochrome b5 between the logarithm of the CYP3A5/CYP3A4 ratios of were correlated with the Km, Vmax, and Vmax/Km Km, Vmax, and Vmax/Km values and the differences between values for CYP3A4 or CYP3A5 (18). In our U values for CYP3A4 and CYP3A5 for steroid hormones.

336 J Pharm Pharm Sci (www.cspsCanada.org) 22, 332 - 339, 2019 previous study, the primary sequence of CYP3A5 CYP3A5 were different from those for CYP3A4, was aligned with that of CYP3A4 using three- and molecular docking simulations might partially dimensional modeling software (18), while in the explain the differences in the affinity of substances present study, we used the recently reported crystal for CYP3A4 and CYP3A5, based on the structures of CYP3A5 (Protein Data Bank code accessibility of substrates to the heme moiety of 6MJM) (25). Vincristine, a typical substrate of CYP3A molecules.

CYP3A5, but not of CYP3A4, has higher Vmax and

Vmax/Km values and a lower Km value for reactions ACKNOWLEDGEMENTS mediated by CYP3A5 than by CYP3A4 (27). In the present study, vincristine was shown to dock closer We are grateful to Motohiro Yasumura for his help to the heme region of CYP3A5 (10.81Å) with a on docking simulation. lower U value than to that of CYP3A4 (18.50Å); the findings were similar to the previous in silico DECLARATION OF INTEREST analysis (18). In the case of midazolam, for which

Km value for CYP3A5 was higher than for CYP3A4, The authors report no conflicts of interest. The the docking energies for CYP3A4 were lower than authors alone are responsible for the content and those for CYP3A5. Although U values for CYP3A5 writing of the paper. in steroid hormones, with the exception of estrone, were lower than those for CYP3A4, the difference in REFERENCES

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